Neuropeptide Y protects rat cortical neurons against β-amyloid toxicity and re-establishes synthesis and release of nerve growth factor.
ABSTRACT: Neuropeptide Y (NPY) is a 36 amino acid peptide, widely distributed within central nervous system neurons. More recently, it has been shown that NPY is involved in Alzheimer's disease (AD), a disorder characterized by accumulation of amyloid β-peptide (Aβ) in neurons. In a previous study, we investigated the effect of NPY on neuronal damage by exposing SH-SY5Y cells (an established human derived neuroblastoma cell line) to Aβ's pathogenic fragment 25-35 (Aβ(25-35)). We found a NPY-neuroprotective action associated with changes in intracellular production of nerve growth factor (NGF), a member of the neurotrophin family. Since our results were encouraging, we decided to replicate our data using primary cortical neurons cultured in presence of Aβ(25-35), and investigated whether NPY had similar neuroprotective action. Moreover, since cortical neurons are able to produce and release NGF, we investigated whether the synthesis and release of NGF were modified in such experimental conditions. Our results showed that a preincubation with NPY counteracted the toxic effect of Aβ, as measured by increased cell viability. Moreover, NPY pretreatment had an effect on NGF since its intracellular synthesis was increased, release was normalized, and mRNA expression was downregulated. Notably, these effects on NGF were in the opposite direction of those produced by incubating the cells with Aβ alone. This study in primary cortical neurons supports the hypothesis that NPY may be a neuroprotective agent against β-amyloid neurotoxicity. These data also suggest that NPY may influence the synthesis and the release of NGF by cortical neurons.
Project description:Activation of the complement cascade, a powerful effector mechanism of the innate immune system, is associated with neuroinflammation but also with elimination of inappropriate synapses during development. Synthesis of C1q, a recognition component of the complement system, occurs in brain during ischemia/reperfusion and Alzheimer's disease, suggesting that C1q may be a response to injury. In vitro, C1q, in the absence of other complement proteins, improves neuronal viability and neurite outgrowth and prevents ?-amyloid-induced neuronal death, suggesting that C1q may have a direct neuroprotective role. Here, investigating the molecular basis for this neuroprotection in vitro, addition of C1q to rat primary cortical neurons significantly upregulated expression of genes associated with cholesterol metabolism, such as cholesterol-25-hydroxylase and insulin induced gene 2, and transiently decreased cholesterol levels in neurons, known to facilitate neurite outgrowth. In addition, the expression of syntaxin-3 and its functional association with synaptosomal-associated protein 25 was increased. C1q also increased the nuclear translocation of cAMP response element-binding protein and CCAAT/enhancer-binding protein-? (C/EBP-?), two transcription factors involved in nerve growth factor (NGF) expression and downregulated specific microRNAs, including let-7c that is predicted to target (and thus inhibit) NGF and neurotrophin-3 (NT-3) mRNA. Accordingly, C1q increased expression of NGF and NT-3, and small interfering RNA inhibition of C/EBP-?, NGF, or NT-3 expression prevented the C1q-dependent neurite outgrowth. No such neuroprotective effect is seen in the presence of C3a or C5a. Finally, the induced neuronal gene expression required conformationally intact C1q. These results show that C1q can directly promote neuronal survival, thereby demonstrating new interactions between immune proteins and neuronal cells that may facilitate neuroprotection.
Project description:Amyloid β protein (Aβ) is closely related to the progression of Alzheimer's disease because senile plaques consisting of Aβ cause synaptic depression and synaptic abnormalities. In the central nervous system, astrocytes are a major glial cell type that contribute to the modulation of synaptic transmission and synaptogenesis. In this study, we examined whether astrocytes exposed to Aβ fragment 25-35 (Aβ25-35) affect synaptic transmission. We show that synaptic transmission by hippocampal neurons was inhibited by astrocytes exposed to Aβ25-35. The Aβ25-35-exposed astrocytes lowered excitatory postsynaptic release and the size of the readily releasable synaptic pool. The number of excitatory synapses was also reduced. However, the number of excitatory synapses was unchanged unless there was direct contact between Aβ25-35-exposed astrocytes and hippocampal neurons. These data indicate that direct contact between Aβ25-35-exposed astrocytes and neurons is critical for inhibiting synaptic transmission in the progression of Alzheimer's disease.
Project description:Neurotrophins, activating the PI3K/Akt signaling pathway, control neuronal survival and plasticity. Alterations in NGF, BDNF, IGF-1, or insulin signaling are implicated in the pathogenesis of Alzheimer disease. We have previously characterized a bigenic PS1×APP transgenic mouse displaying early hippocampal Aβ deposition (3 to 4 months) but late (17 to 18 months) neurodegeneration of pyramidal cells, paralleled to the accumulation of soluble Aβ oligomers. We hypothesized that PI3K/Akt/GSK-3β signaling pathway could be involved in this apparent age-dependent neuroprotective/neurodegenerative status. In fact, our data demonstrated that, as compared with age-matched nontransgenic controls, the Ser-9 phosphorylation of GSK-3β was increased in the 6-month PS1×APP hippocampus, whereas in aged PS1×APP animals (18 months), GSK-3β phosphorylation levels displayed a marked decrease. Using N2a and primary neuronal cell cultures, we demonstrated that soluble amyloid precursor protein-α (sAPPα), the predominant APP-derived fragment in young PS1×APP mice, acting through IGF-1 and/or insulin receptors, activated the PI3K/Akt pathway, phosphorylated the GSK-3β activity, and in consequence, exerted a neuroprotective action. On the contrary, several oligomeric Aβ forms, present in the soluble fractions of aged PS1×APP mice, inhibited the induced phosphorylation of Akt/GSK-3β and decreased the neuronal survival. Furthermore, synthetic Aβ oligomers blocked the effect mediated by different neurotrophins (NGF, BDNF, insulin, and IGF-1) and sAPPα, displaying high selectivity for NGF. In conclusion, the age-dependent appearance of APP-derived soluble factors modulated the PI3K/Akt/GSK-3β signaling pathway through the major neurotrophin receptors. sAPPα stimulated and Aβ oligomers blocked the prosurvival signaling. Our data might provide insights into the selective vulnerability of specific neuronal groups in Alzheimer disease.
Project description:Alzheimer disease (AD), a progressive neurodegenerative disorder, is characterized by cognitive decline and the accumulation of senile plaques in the brain. Amyloid β protein (Aβ) in the plaques is thought to be responsible for the memory loss in AD patients. [Gly14]-humanin (HNG), a derivative of humanin (HN), has much stronger neuroprotective effects than natural HN in vitro. However, clarification of the Aβ active center and the neuroprotective mechanism of HN still need in vivo evidence. The present study first compared the in vivo biological effects of three Aβ fragments (1-42, 31-35, and 35-31) on spatial memory in rats, and investigated the neuroprotective effects and molecular mechanisms of HNG. The results showed that intrahippocampal injection of Aβ1-42 and Aβ31-35 almost equally impaired spatial learning and memory, but the reversed sequence Aβ35-31 did not have any effect; a high dose of Aβ31-35 (20 nmol) produced a more detrimental response than a low dose (2 nmol); Aβ31-35 injection also disrupted gene and protein expression in the hippocampus, with up-regulation of caspase3 and down-regulation of STAT3; pretreatment with HNG not only protected spatial memory but also rescued STAT3 from Aβ-induced disruption; and the neuroprotective effects of HNG were effectively counteracted by genistein, a specific tyrosine kinase inhibitor. These results clearly show that sequence 31-35 in Aβ is the shortest active center responsible for the neurotoxicity of Aβ from molecule to behavior; and HNG protects spatial learning and memory in rats against Aβ-induced insults; and probably involves the activation of tyrosine kinases and subsequent beneficial modulation of STAT3 and caspase3.
Project description:It is generally accepted that the amyloid ? (A?) peptide toxicity contributes to neuronal loss and is involved in the initiation and progression of Alzheimer's disease (AD). Cold-inducible RNA-binding protein (CIRBP) is reported to be a general stress-response protein, which is induced by different stress conditions. Previous reports have shown the neuroprotective effects of CIRBP through the suppression of apoptosis via the Akt and ERK pathways. The objective of this study is to examine the effect of CIRBP against A?-induced toxicity in cultured rat primary cortical neurons and attempt to uncover its underlying mechanism. Here, MTT, LDH release, and TUNEL assays showed that CIRBP overexpression protected against both intracellular amyloid ?- (iA?-) induced and A? 25-35-induced cytotoxicity in rat primary cortical neurons. Electrophysiological changes responsible for iA?-induced neuronal toxicity, including an increase in neuronal resting membrane potentials and a decrease in K+ currents, were reversed by CIRBP overexpression. Western blot results further showed that A? 25-35 treatment significantly increased the level of proapoptotic protein Bax, cleaved caspase-3, and cleaved caspase-9 and decreased the level of antiapoptotic factor Bcl-2, but were rescued by CIRBP overexpression. Furthermore, CIRBP overexpression prevented the elevation of ROS induced by A? 25-35 treatment by decreasing the activities of oxidative biomarker and increasing the activities of key enzymes in antioxidant system. Taken together, our findings suggested that CIRBP exerted protective effects against neuronal amyloid toxicity via antioxidative and antiapoptotic pathways, which may provide a promising candidate for amyloid-based AD prevention or therapy.
Project description:Histological and morphological studies indicate that approximately 5% of striatal neurons are cholinergic or ?-aminobutyric acidergic (GABAergic) interneurons (gINs). However, the number of striatal neurons expressing known interneuron markers is too small to account for the entire interneuron population. We therefore studied the serotonin (5HT) receptor 3a-enhanced green fluorescent protein (5HT3a(EGFP)) mouse, in which we found that a large number of striatal gINs are labeled. Roughly 20% of 5HT3a(EGFP)-positive cells co-express parvalbumin and exhibit fast-spiking (FS) electrophysiological properties. However, the majority of labeled neurons do not overlap with known molecular interneuron markers. Intrinsic electrical properties reveal at least 2 distinct novel subtypes: a late-spiking (LS) neuropeptide-Y (NPY)-negative neurogliaform (NGF) interneuron, and a large heterogeneous population with several features resembling low-threshold-spiking (LTS) interneurons that do not express somatostatin, NPY, or neuronal nitric oxide synthase. Although the 5HT3a(EGFP) NGF and LTS-like interneurons have electrophysiological properties similar to previously described populations, they are pharmacologically distinct. In direct contrast to previously described NPY(+) LTS and NGF cells, LTS-like 5HT3a(EGFP) cells show robust responses to nicotine administration, while the 5HT3a(EGFP) NGF cell type shows little or no response. By constructing a molecular map of the overlap between these novel populations and existing interneuron populations, we are able to reconcile the morphological and molecular estimates of striatal interneuron numbers.
Project description:Acetylcholine (ACh) synthesis and release from basal forebrain cholinergic neurons (BFCN) innervating the cerebral cortex and hippocampus are essential processes for normal learning, memory and attention. Bone morphogenetic protein (BMP) 9 is a cholinergic differentiation factor in the developing septum that increases ACh synthesis and choline acetyltransferase (Chat) gene expression both in vivo and in vitro. We investigated the possible induction of cholinergic trophic factors by BMP9 in murine septal cells. Nerve growth factor (NGF) protein expression and secretion into the medium was increased in cultured embryonic septal cells treated with BMP9, and partially mediated BMP9-induced acetylcholine production and Chat gene expression. BMP9-induced Ngf gene expression was detected in postmitotic cells, required new protein synthesis and was blocked by BMP type I receptor inhibition. Cholinergic neurons were isolated by fluorescence-activated cell sorting based on either transgenic expression of green fluorescent protein driven by the Chat promoter or NGF receptor (p75) immunostaining. Although both noncholinergic and cholinergic neurons in untreated cultures expressed similar low levels of Ngf, increased Ngf gene expression was restricted to Chat-positive neurons in BMP9-treated cultures. Likewise, similar levels of Ngf mRNA were detected in p75-negative and p75-positive septal cells, yet only p75-positive BFCN increased their Ngf gene expression when treated with BMP9, and only these cells expressed the Alk1 BMP receptor. The data suggest an autocrine/paracrine role for NGF in the development and/or maintenance of BFCN and imply that the stimulation of NGF production and release contributes to the cholinergic-supportive properties of BMP9.
Project description:Despite the central role of amyloid β (Aβ) peptide in the etiopathogenesis of Alzheimer's disease (AD), its physiological function in healthy brain is still debated. It is well established that elevated levels of Aβ induce synaptic depression and dismantling, connected with neurotoxicity and neuronal loss. Growing evidence suggests a positive regulatory effect of Aβ on synaptic function and cognition; however the exact cellular and molecular correlates are still unclear. In this work, we tested the effect of physiological concentrations of Aβ species of endogenous origin on neurotransmitter release in rat cortical and hippocampal neurons grown in dissociated cultures. Modulation of production and degradation of the endogenous Aβ species as well as applications of the synthetic rodent Aβ40 and Aβ42 affected efficacy of neurotransmitter release from individual presynapses. Low picomolar Aβ40 and Aβ42 increased, while Aβ depletion or application of low micromolar concentration decreased synaptic vesicle recycling, showing a hormetic effect of Aβ on neurotransmitter release. These Aβ-mediated modulations required functional alpha7 acetylcholine receptors as well as extracellular and intracellular calcium, involved regulation of CDK5 and calcineurin signaling and increased recycling of synaptic vesicles. These data indicate that Aβ regulates neurotransmitter release from presynapse and suggest that failure of the normal physiological function of Aβ in the fine-tuning of SV cycling could disrupt synaptic function and homeostasis, which would, eventually, lead to cognitive decline and neurodegeneration.
Project description:Neuropeptide Y (NPY) is an abundant neuropeptide of the neocortex involved in numerous physiological and pathological processes. Because of the large electrophysiological, molecular, and morphological diversity of NPY-expressing neurons their precise identity remains unclear. To define distinct populations of NPY neurons we characterized, in acute slices of rat barrel cortex, 200 cortical neurons of layers I-IV by means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reverse transcriptase-PCR designed to probe for the expression of well established molecular markers for cortical neurons. To classify reliably cortical NPY neurons, we used and compared different unsupervised clustering algorithms based on laminar location and electrophysiological and molecular properties. These classification schemes confirmed that NPY neurons are nearly exclusively GABAergic and consistently disclosed three main types of NPY-expressing interneurons. (1) Neurogliaform-like neurons exhibiting a dense axonal arbor, were the most frequent and superficial, and substantially expressed the neuronal isoform of nitric oxide synthase. (2) Martinotti-like cells characterized by an ascending axon ramifying in layer I coexpressed somatostatin and were the most excitable type. (3) Among fast-spiking and parvalbumin-positive basket cells, NPY expression was correlated with pronounced spike latency. By clarifying the diversity of cortical NPY neurons, this study establishes a basis for future investigations aiming at elucidating their physiological roles.
Project description:The transcellular signaling of neurotrophins is postulated, but evidence is scarce. We now show that a small number of NT4- and BDNF-overexpressing neurons in the cortical explant of thalamocortical cocultures rapidly evoked a Trk receptor-dependent upregulation of neuropeptide Y (NPY) mRNA in interneurons. In contrast to BDNF, the action of NT4 was independent of calcium influx through NMDA receptors and L-type calcium channels. NPY neurons vastly outnumbered the neurotrophin-overexpressing neurons (mostly pyramidal cells), arguing for a spread of the neurotrophin signal via axonally connected neuronal populations. Furthermore, NT4 transfection of one explant of axonally connected corticocortical cocultures evoked significantly larger numbers of NPY neurons in both explants. Delivery of the signal was not by diffusion of neurotrophins via the medium. Moreover, cortical NPY neuron numbers increased after NT4 and BDNF transfection of a cocultured tectal explant innervated selectively by cortical layer V pyramidal neurons. The transcellular induction of NPY suggests a source-to-sink model for axonal transport and a local cortical redistribution of TrkB ligands to interneurons competent for NPY expression.